论文部分内容阅读
目的 探讨5-氮杂-2’-脱氧胞苷(5-Aza-dC)联合曲古抑菌素A(TSA)对分化程度不同的人胃腺癌细胞株MKN-45、SGC-7901与MGC-803的细胞存活率和抑癌基因E-cadherin启动子区甲基化以及mRNA表达的影响. 方法 培养3个细胞株,实验分为空白对照组、5-Aza-dC组(10μmol/L 5-Aza-dC处理72 h)、TSA组(1μmol/L TSA处理48 h)和联合组(10μmol/L5-Aza-dC处理24 h后,再加入1μmol/L TSA处理48 h).观察三株细胞形态的改变,用台盼蓝染色法检测活细胞数.采用甲基化特异性聚合酶链反应(MSP)和反转录聚合酶链反应(RT-PCR)分别检测3株细胞的E-cadherin基因启动子区甲基化水平与mRNA表达. 结果 MKN-45细胞中,5-Aza-dC组、TSA组与联合组的细胞存活率分别为(73.03±1.92)％、(64.48±1.28)％和(52.44±1.45)％;SGC-7901细胞分别是(63.33±1.92)％、(52.15±1.44)％和(44.44±1.45)％;MGC-803细胞分别是(57.33±1.61)％、(45.48±1.29)％和(33.11±1.12)％.3株细胞的单药组的细胞存活率均减少,联合组分别比5-Aza-dC和TSA处理组明显下降(均P＜0.05).E-cadherin基因启动子区甲基化水平,5-Aza-dC组、TSA组及联合组的甲基化产物与空白对照组相比较均降低,其中联合组比单药组降低明显.空白对照、5-Aza-dC、TSA及二者联合处理组的E-cadherin的mRNA相对表达量在MKN-45细胞株中分别为0.17±0.01、0.32±0.01、0.29±0.02和0.57±0.02;在SGC-7901细胞株中分别为0.22±0.01、0.46±0.01、0.43±0.01和0.61±0.01;在MGC-803细胞株中分别为0.24±0.02、0.49±0.01、0.44±0.01和0.73±0.01.在单药处理组中比空白对照组升高(P＜0.05),并且二药联合组比单药组升高(P＜0.01). 结论 5-Aza-dC和TSA单独处理均降低分化程度不同的胃癌细胞存活率,使E-cadherin基因的甲基化水平下降,并增加其mRNA表达.3株细胞的联合组的影响效果更明显,两药之间具有协同作用.其中MKN-45的存活率,甲基化水平和mRNA表达的改变均较SGC-7901与MGC-803明显.“,”Objective To observe the effects of 5-aza-2 ’-deoxycytidine (5-Aza-dC) and trichostatin A (TSA) alone and combination on cell survival rate and promoter methylation and mRNA expression level of tumor suppressor gene E-cadherin in three human gastric cancer cell lines MKN-45,SGC-7901 and MGC-803 with different differentiation degree.Methods MKN-45,SGC 7901 and MGC-803 cells were routinely cultured,and were divided into blank control,5-Aza-dC (10μmol/L for 72 h),TSA (1 μmol/L for 48h) and the combination (10μmol/L 5-Aza-dC for 72 h plus 1 μmol/L TSA for last 48 hours) groups.Morphological changes of the gastric cells were observed.The number of live cells was detected by trypan blue staining,and the cell survival rate was calculated.The promoter methylation and mRNA expression of E-cadherin gene were detected by methylation-specific PCR (MSP) and RT-PCR methods,respectively.Results The survival rates in groups of 5-Aza-dC,TSA and combination were (73.03±1.92)％,(64.48±1.28)％ and (52.44±1.45)％ for MKN-45 cells,and (63.33±1.92)％,(52.15±1.44)％ and (44.44±1.45)％ for SGC-7901 cells,and (57.33±1.61)％,(45.48±1.29)％ and (33.11±1.12)％ for MGC-803 cells.It was more significant that the cell survival rates in the three cell lines were significant decreased in the combination group than that in single drug group (all P＜0.05).The promoter methylation level of E-cadherin gene were lower in each drug group than that in blank control group,and the combination group showed more remarkable decrease than single drug groups.The relative mRNA expression of E-cadherin in control,5-Aza-dC,TSA and combination groups was (0.17 ± 0.01),(0.32 ± 0.01),(0.29±0.02) and (0.57±-0.02) in MKN-45 cells,(0.22±0.01),(0.46±0.01),(0.43±0.01)and (0.61±0.01) in SGC-7901 cells,(0.24±0.02),(0.49±0.01),(0.44±0.01) and (0.73±0.01) in MGC-803 cells,respectively.There was significant decrease of E-cadherin mRNA level in single drug groups,as compared with blank control group,and E-cadherin mRNA level was more decreased in combination group than that in single-drug groups.Conclusions The 5-Aza-dC and TSA single-drugs treatment can alone reduce the cell survival rate in the three gastric cancer cell lines and E-cadherin gene methylation,and increase the expression of E-cadherin mRNA,and the 5-Aza-dC and TSA combination treatment has a more potent efficacy and synergistic effect.The changes in survival rates and promoter methylation and mRNA levels of E-cadherin gene are more obvious in cell line MKN-45 than in cell lines SGC-7901 and MGC-803.Our results suggest that 5-Aza-dC and TSA have potentials used in the clinical treatment of gastric cancer.